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Neural and Hormonal Control of Expression of Myogenic
Regulatory Factor Genes During Regeneration of Xenopus
Fast Muscles: Myogenin and MRF4 mRNA Accumulation
Are Neurally Regulated Oppositely
Laboratoire de Biologie du Développement et de la Différenciation Musculaire, Paris, France
Laboratoire de Neurobiologie; Université René Descartes, Paris, France
With the aim to investigate the
influence of both innervation and thyroid hormone, on the expression of the MRFs during muscle regeneration, we performed cardiotoxin injury-induced regeneration experiments on fast
muscles of adult Xenopus laevis subjected to different experimental conditions, including denervation and T3 treatment, and analyzed the accumulation of the four myogenic regulatory factors
(MRFs) using RT-PCR and in situ hybridization.
We show here that manipulation of hormone levels or innervation resulted in differential alterations of MRF expression. Denervation and T3
treatment transiently down-regulated Myf-5
mRNA levels at the beginning of the regeneration process. Myf-5 was the only myogenic factor
subject to thyroid hormone influence. Muscle denervation persistently reduces the levels of
MRF4 transcripts as early as the first stages of
regeneration, whereas the levels of myogenin
mRNA were increased in the late stages of regeneration. This suggests that MRF4 expression may
be induced by innervation and hence may be involved in mediating transcriptional responses to
innervation and that myogenin expression may
compensate for the down-regulation of MRF4
gene. This switch in MRF gene expression following denervation could have important consequences for the ability of Xenopus regenerating
muscles to recover function after denervation.
Dev Dyn 2000;218:112–122. © 2000 Wiley-Liss, Inc.
Key words: muscle regeneration; myogenic regulatory factors; innervation; thyroid
hormone; Xenopus
The four myogenic regulatory factors (MRFs): MyoD
(Davis et al., 1987), Myf-5 (Braun et al., 1989), myogenin (Wright et al., 1989), and MRF4 (Rhodes and
Konieczny, 1989; Braun et al., 1990; Miner and Wold,
1990), are basic helix-loop-helix transcription factors
whose ectopic expression is able to convert a wide
range of cultured cells to a muscle phenotype (Schäfer
et al., 1990; Choi et al., 1990) and which can promote
the transcription of a number of muscle-specific genes
(Weintraub et al., 1991). The functions of the MRFs in
vivo have been investigated by determining the pattern
of their expression, by gene targeting and ectopic expression. Gene knock-out mice experiments have elucidated the hierarchical relationships existing between
the MRFs. MyoD and Myf-5 are required for the determination of skeletal myoblasts, and myogenin and
MRF4, act later in the program, probably as differentiation factors (reviewed in Buckingham, 1994; Ludolph and Konieczny, 1995). During development, the
order of expression of MRF genes varies according to
muscle origin and between species. In Xenopus, the key
difference lies in the observation that XMyoD and
XMyf-5 mRNA can be detected in presomitic mesoderm
(Hopwood et al., 1989; 1991), which is in contrast to the
murine system. Moreover, myogenin transcript was
only detected during secondary myogenesis of Xenopus
and never during somitogenesis (Jennings, 1992; Nicolas et al., 1996, 1998a).
An important feature of mature skeletal muscles is
their ability to regenerate following injury. Satellite
cells, closely associated with muscle fibers, are myoblast-like cells responsible for the regenerative capacity of muscles (Campion, 1984). These adult muscle
stem cells are normally mitotically quiescent but are
activated in response to injury. The regenerative process is characterized by the proliferation of the descendants of the activated satellite cells, called myogenic
precursor cells (mpc), before fusing to form new myotubes which differentiate into mature myofibers. Given
the importance of the MRFs for myoblast differentiation during development, the pattern of expression of
the MRFs has been analyzed during muscle regeneration in mammals (Grounds et al., 1992; Füchtbauer et
al., 1992; Kami et al., 1995; Rantanen et al., 1995) and
amphibians (Nicolas et al., 1996, 1998b) following different types of injury. However, little is known about
*Correspondence to: Pr. C. Chanoine, Laboratoire de Biologie du
Développement et de la Différenciation Musculaire (EA 2507), Centre
Universitaire des Saints-Péres, Université René Descartes, 45 rue des
Saints-Péres, 75270 Paris cedex 06, France.
Received 13 September 1999; Accepted 31 Janaury 2000
Fig. 2. RNAse protection for acetylcholine receptor (␣ subunit) and
EF-1␣ RNA. Amounts used were 5 ␮g total RNA per assay; innervated
(C) and denervated (D) regenerating muscles taken at 15 days following
cardiotoxin injury.
Fig. 1. Analysis of serum thyroid hormone levels in control adult
animal (C), T3 treated adult animal (T3) and prometamorphic larvae (M).
the specific involvement of innervation and thyroid
hormone in the accumulation of the MRF transcripts
during muscle regeneration (Koishi et al., 1995). Yet, it
is well known that these two factors have a critical role
in determining adult muscle phenotype. It is also well
established that embryonic myoblasts and satellite
cells are not equivalent cells (Stockdale, 1992) which
are submitted to distinct neural and hormonal environment. Moreover innervation and thyroid hormone are
able to regulate MRF expression in vitro (reviewed in
Muscat et al., 1995), and are also controlling factors in
adult muscles (Hughes et al., 1993; Adams et al., 1995).
Therefore, these epigenetic factors could be involved in
MRF expression following muscle injury in adult animals.
In this work, to investigate the influence of both
innervation and thyroid hormone on the expression of
the MRFs during muscle regeneration, we performed
cardiotoxin injury-induced regeneration experiments
on fast muscles of adult Xenopus laevis subjected to
different experimental conditions, including denervation and T3 treatment, and analyzed the accumulation
of MyoD, Myf-5, myogenin, and MRF4 using RT-PCR
and in situ hybridization.
We show here that manipulation of hormone levels
or innervation resulted in alterations of MRF expression. These results are discussed in relation to the
ability of Xenopus regenerating muscles to recover
function following epigenetic changes.
The sequence of histological changes as well as the
pattern of expression of MRFs observed in Xenopus
regenerating muscle following cardiotoxin injury has
been previously described (Saadi et al., 1994; Nicolas et
al., 1996, 1998b). A single injection of cardiotoxin
caused an almost complete degeneration of the myofi-
bers within 24 hr. In the present study, regenerative
stages corresponded to the following days: mononucleate cells/young myotubes, 11 days postinjection; young
myotubes, 15 days postinjection; large myotubes, 20
days postinjection; and mature myofibers, 1 month
postinjection. To be sure of the completeness of the
degeneration/ regeneration process, all muscle samples
were monitored as indicated in Experimental Procedures. To analyze the accumulation of MRF transcripts
during muscle regeneration, we chose to use both in
situ hybridization and a semi-quantitative RT-PCR assay able to detect small amounts of transcripts and
thus offering the opportunity to analyze small samples
(Nicolas et al., 1998a). The analyzed transcripts were
co-reverse-transcripted and co-amplified in the same
reaction with the ubiquitously expressed EF-1␣ gene
(Krieg et al., 1989) to serve as internal control for the
amount of RNA tested and for the RT-PCR reproducibility.
Thyroid hormone is required for the normal development of the central nervous system (Dussault and
Ruel, 1987) and probably peripheral nerves, which suggested that the regulation by thyroid hormone of some
muscle-specific genes could be nerve-mediated. To determine whether T3 acts via the nerve innervating the
brachial muscle, we removed a portion of one of the two
brachial nerve in adult Xenopus just before cardiotoxin
injection in the two anterior brachial muscles of the
same animal. One set was treated with T3 and another
set was untreated. The innervated contralateral muscles of the operated animals were compared to the
denervated muscles since both should be exposed to the
same circulating levels of T3, thus providing a better
control for this parameter than independently T3
treated Xenopus. The hyperthyroidian status of the
T3-treated animals was monitored by measuring the
circulating T3 levels (Fig. 1). In denervated regenerating muscles, AChR ␣ subunit RNA was strongly induced, confirming that denervation was successful,
while another control RNA, EF-1␣ (Krieg et al, 1989),
was unaffected by denervation (Fig. 2 and Jennings,
1992). At the first stage of muscle regeneration analyzed (11 days P-I), semi-quantitative RT-PCR showed
a strong decrease of Myf-5 mRNA levels as much as
3– 4-fold, following denervation as well as T3 treatment
Fig. 3. Semi-quantitative analysis of MRF expression at different
stages of regeneration (11 days, 15 days, 20 days, and 30 days P-I) and
in different experimental conditions assayed by RT-PCR amplification. A:
Myf-5, B: MyoD, C: MRF4, and D: myogenin. Intensity of each PCR
product signals was quantified after scanning with the NIH Image analyzer software. The MRF signal was normalized to EF-1␣ signal, and the
relative amount of each sample PCR product is presented. Five distinct
RT-PCR analysis for one sample were performed. *significantly different
from C value, P ⬍ 0.05. C, natural hypothyroidism; T3, T3 treatment; D,
denervation; DT3, T3 treatment and denervation.
(Fig. 3A). This down-regulation of Myf-5 gene by our
experimental treatments was confirmed by in situ hybridization (Fig. 4). The fact that T3 treatment induced
a similar effect on Myf-5 gene expression in innervated
as well as in denervated regenerating muscles showed
that T3 acts independently of innervation. This demonstrated that muscle denervation and T3 treatment
were involved in the down-regulation of Myf-5 gene
expression in two distinct ways. From day 15 P-I, both
denervation and/or T3 treatment have no significant
effects on the expression of Myf-5 (Fig. 3A). Semi-quantitative RT-PCR revealed that, at all stages of the
regenerating process, neither denervation nor T3 treatment modified the MyoD mRNA levels (Fig. 3B).
From 11 days P-I to 1 month P-I, RT-PCR analysis
showed that MRF4 mRNA was significantly downregulated by denervation whereas T3 treatment had
no effect on the accumulation of this MRF mRNA
(Fig. 3C). In contrast to what was observed for
MRF4, muscle denervation increases the levels of
myogenin mRNA in the late stages of muscle regeneration, from 20 days P-I (Fig. 3D). These results
were confirmed by in situ hybridization: as early as
the first stage of regeneration (11 days P-I), the
hybridization signal for MRF4 transcripts was significantly reduced in denervated muscles in comparison to that observed in innervated contralateral
muscles as well as in innervated muscles of T3treated animals (Fig. 5). This fall of the level of
MRF4 mRNA due to muscle denervation was continuously detected in the following stages of regeneration as shown in large myotubes at 20 days P-I (Fig.
6). In situ hybridization experiments also clearly
confirmed that T3-treatment has no effect on the
accumulation of MRF4 mRNAs during muscle regeneration (Fig. 5C and D; Fig. 6D and E). For myogenin
transcripts, as early as the large myotube stage, the
hybridization signal detected in denervated muscles
of control as well as T3-treated animals was strong in
comparison to innervated contralateral muscles
where myogenin transcripts were not detected using
in situ hybridization, in accordance with our previous results (Nicolas et al., 1996) (Fig. 7). It should be
noted that the response of muscle to denervation is
more belated in the case of myogenin in comparison
to that of MRF4: the down-regulation of MRF4 is
observed as early as the beginning of the regenerating process, whereas the up-regulation of the myogenin gene only appears in the late stages of muscle
regeneration (Fig. 3 D). Like that observed for MRF4,
the accumulation of myogenin transcripts is unaffected by T3 treatment (Figs. 3D, 7).
This article provides a detailed analysis of both the
neural and the hormonal influences on the expression
of the MRF genes during regeneration of Xenopus fast
muscles. It shows that in Xenopus, the accumulation of
each MRF mRNA is differentially regulated by innervation and/or thyroid hormone during in vivo myogenesis. Thus, each member of the MyoD family may have
a distinct role in the maintenance of the specific physiological properties of Xenopus muscle.
Fig. 4. In situ hybridization using antisense riboprobes to Myf-5 on
transverse sections of control (A, D), T3-treated (B) and denervated (C)
regenerating muscle at 11 days P-I. Using sense riboprobes, we did not
detect hybridization signals (data not shown). A, B and C are darkfield
photomicrographs. D is a brightfield photomicrograph of the same section
that is shown in A. Scale bar ⫽ 200 ␮m
Myf-5 Is the Only MRF Subject to Hormonal
Regulation During Muscle Regeneration of
mRNA in slow twitch soleus fibers (Hughes et al., 1993).
From these results, it has been hypothetized that, at the
level of the muscle cells, one or several members of the
MyoD family could mediate the effects of TH on some
contractile protein genes (Muscat et al., 1995). In Xenopus, the correlative accumulation of myogenin and fast
MHC mRNAs in secondary myofibers, when TH levels
increase during metamorphosis, had also suggested this
type of regulation (Nicolas et al., 1998a). Surprisingly, in
the present study, we find that T3 treatment does not
up-regulate any of the four MRF mRNA levels. In contrast, Myf-5 transcripts were down-regulated by T3 in the
first step of the regeneration process. To our knowledge,
this down-regulation of Myf-5 by TH has never been
described in any in vitro or in vivo models and should be
considered in relation to the potential involvement of
Myf-5 at the beginning of myogenesis.
It appears that during three types of myogenesis in
Xenopus, including somitogenesis, regeneration and limb
It is known that thyroid hormone is a major controlling
influence of myogenesis. T3 regulates the expression of
many muscle-specific genes (d’Albis et al., 1987;
Chanoine et al., 1987, 1989; Saadi et al., 1993). It has
been observed that T3 treatment of the myogenic cell line,
C2.7, promoted terminal differentiation, increased MyoD
gene transcription and resulted in the precocious expression of myogenin and contractile protein mRNAs (Carnac
et al., 1992). Furthermore, it was observed that overexpression of the c-erbA ␤ gene encoding thyroid hormone
receptor (TR) in myogenic cells enhanced differentiation
and increased MyoD expression in the presence of T3
(Begue et al., 1993). In the rat, it has also been demonstrated that MyoD selectively accumulates in fast twitch
fiber types and that thyroid hormone treatment results in
the significant induction of MyoD and fast IIa MHC
Fig. 5. In situ hybridization using antisense riboprobes to MRF4 on
transverse sections of control (C), T3-treated (D) and denervated (A,B)
regenerating muscle at 11 days P-I. Using sense riboprobes, we did not
detect hybridization signals (data not shown). B, C and D are darkfield
photomicrographs. A is a brightfield photomicrograph of the same section
that is shown in B. Scale bar ⫽ 200 ␮m
development (Hopwood et al., 1989, 1991; Rupp and
Weintraub, 1991; Nicolas et al., 1996, 1998a and b),
MyoD and Myf-5 are the first myogenic factors expressed.
Microinjection of synthetic XMyf-5 or XMyoD mRNA into
early embryos suggest that XMyf-5 might act largely
during the very early stages of myogenesis before MyoD.
It is also interesting to note that the XMyf-5 gene is only
transiently expressed in all types of myogenesis in Xenopus whereas XMyoD is continuously expressed, XMyoD
mRNA being the major MRF transcript detected in Xenopus adult muscle (Jennings, 1992).
In tissue culture, the differentiation of myoblasts is
tightly regulated through a suppression mechanism
mediated by a gamut of oncogene products and growth
factors which prevent cell cycle arrest and repress
trans-activation of myogenic gene expression. Hormonal stimulation (thyroid hormone, retinoic acid (RA)
and insulin-like growth factors (IGFs)) and growth factor deprivation induce proliferating myoblasts to exit
the cell cycle and fuse into post-mitotic multinucleated
myofibers that express muscle-specific phenotypic
markers (rev. in Muscat et al., 1995). In particular, it
has been observed that all-trans and 9-cis RA repress
the Myf-5 gene at the transcriptional level (Carnac et
al., 1993). We can suggest that the increase of circulating T3 levels in TH-treated animals forced the proliferating myoblasts to differentiate, involving the downregulation of Myf-5 gene and the expression to musclestructural genes. We have recently shown that T3 upregulated the transcripts coding for the fast MHC as
early as the first stages of muscle regeneration (unpublished results).
Myogenin and MRF4 mRNA Accumulation Are
Neurally Regulated Oppositely
It is known that innervation induces many changes
in muscle gene expression (reviewed in Laufer and
Changeux, 1989). Many of these are caused by electri-
Fig. 6. In situ hybridization using antisense riboprobes to MRF4 on
transverse sections of control (A, C), T3-treated (D), denervated (B), and
denervated/T3-treated (E) regenerating muscle at 20 days P-I. Using
sense riboprobes, we did not detect hybridization signals (data not
shown). B, C, D, and E are darkfield photomicrographs. A is a brightfield
photomicrograph of the same section that is shown in C. A–C: Scale
bar ⫽ 200 ␮m; D, E: Scale bar ⫽ 150 ␮m
cal activity in the muscle, for example the suppression
of acetylcholine receptor expression in extrajunctional
regions of muscle fibers or the expression of particular
MHC isoforms (Goldman et al., 1988; Kirschbaum et
al., 1990). Many studies have shown that the loss of
electrical stimulation can lead to up-regulation of myogenic HLH mRNAs (Eftimie et al., 1991; Piette et al.,
1992; Hughes et al., 1993), suggesting that the myogenic HLH transcription factors could mediate the effects of the nerve. In mammals, Duclert et al. (1991)
showed that MRF4 mRNA as well as myogenin mRNA
were induced by denervation. From the literature, it
seems that these two MRFs also play the central role in
mediating nerve influence on muscle gene expression
(Sunyer et Merlie, 1993; Buonanno et al., 1993; Merlie
et al., 1994).
Our study reveals that myogenin and MRF4 mRNAs
are regulated in two opposite ways by denervation in
contrast to what is observed in mammals (Adams et al.,
1995). We show that muscle denervation persistently
reduces the levels of MRF4 transcripts during regeneration whereas the levels of myogenin mRNA increase
in the late stages of regeneration. Jennings (1992) has
also previously shown that a short-term denervation
Fig. 7. In situ hybridization using antisense riboprobes to myogenin
on transverse sections of control (C, G, H), T3-treated (A, B), denervated
(D, E, I) and denervated/T3-treated (F) regenerating muscle at 20 (A–F)
and 30 (G–I) days P-I. Using sense riboprobes, we did not detect hybridization signals (data not shown). B, C, E, F, H, I are darkfield photomicrographs. A, D, G are brightfield photomicrographs. Scale bar ⫽ 200 ␮m
produces a loss of MRF4 mRNA in uninjured muscle of
adult Xenopus. This continuous down-regulation of
MRF4 gene expression by denervation raises the pos-
sibility that MRF4 expression may be induced by innervation and hence may be involved in mediating
transcriptional responses to innervation. During Xeno-
pus myotome development, MRF4 mRNA is detected
later, after MyoD and Myf-5 transcripts (Jennings,
1992), and its expression overlaps with the formation of
neuromuscular connections (Blackshaw and Warner,
1976; Kullberg et al., 1977). It is interesting to note
that in vitro studies have recently shown that MRF4
and the AchR epsilon-subunit gene, both specifically
expressed in mature adult skeletal muscle of mammals, were found to be coexpressed at the formation of
multinucleated spontaneously contracting myotubes
(Rohwedel et al., 1998). This result and the fact that
MRF4 is the MRF which preferentially transactivates
the epsilon-promoter (Sunyer et Merlie, 1993) reinforce
the possibility of an involvement of MRF4 in the regulation of nerve-regulated genes. The fact that the myogenin gene is up-regulated by denervation suggests
that myogenin expression may compensate for the
down-regulation of the MRF4 gene. It has been also
shown that homozygous null mutant mice for MRF4
(⫺/⫺) exhibit fairly normal muscle and display an approximately threefold increase in myogenin expression,
leaving open the possibility that myogenin also compensates for the loss of MRF4 in these embryos (Zhang
et al., 1995). Nevertheless, it seems clear, in mammals,
that myogenin and MRF4 do not have totally redundant functions, since myogenin (⫺/⫺) mice form myoblasts that fail to form myotubes efficiently in vivo. In
Xenopus, this is not clear but our previous data (Nicolas et al., 1998a and b) point to the same conclusions.
Whereas denervation does not have a consistent effect on MyoD expression in regenerating muscle of
Xenopus, as observed in Xenopus adult muscles (Jennings, 1992), the fact that Myf-5 mRNA is transiently
down-regulated by denervation in the first stage of
regeneration is a surprising result. Indeed, previous
reports indicated that no change or only little change
occurred in the level of Myf-5 mRNA in denervated
muscle of mice (Duclert et al., 1991) and chicken (Saitoh et al., 1993).
The cause of the discrepancy between Xenopus and
mammalian or chicken results concerning MRF expression may reflect a diversity in response pathways to
denervation or thyroid hormone treatment. In Xenopus, specific mechanisms may be responsible for physiological properties that are specific to Xenopus muscle.
In mammals, muscle denervation has a dramatic effect
on muscle morphology. In particular, denervated muscles are characterized by an important atrophy. In
contrast, in Xenopus, denervated muscles did not
present a different histological pattern as compared to
innervated muscles (Jennings, 1992): atrophy of denervated muscle that is observed in mammals, was never
detected in Xenopus.
During regeneration of Xenopus muscle, Myf-5
and/or MyoD, which are expressed in activated satellite cells (Nicolas et al., 1996, 1998b) could be involved
in the early events of the regeneration process. We can
hypothesize that the decrease in Myf-5 mRNA levels
could be compensated by a stable and unaffected MyoD
expression following variation in hormone levels and/or
denervation. Thus, a stable MyoD expression could
contribute to the initial differentiation events and play
a crucial role in initiating the regeneration process as
reported for mice (Megeney et al., 1996). In a second
step, the “differentiation factors,” myogenin and
MRF4, may contribute to the terminal differentiation
events with partially overlapping functions (see above).
Interestingly, gene dosage compensation of “early” or
“late” MRF transcripts seems to follow epigenetic
changes. A decreased expression of the early transcript
Myf-5 is compensated by a stable expression of MyoD.
Similarly, a decreased expression of the late transcript
MRF4 is compensated by an increased expression of
the other late MRF transcript, myogenin. We can speculate that the induction of myogenin gene and possibly
the continuous expression of MyoD transcripts following denervation contribute to maintain the specific
physiological properties of Xenopus muscle.
Adult Xenopus laevis were maintained at 22°C in tap
water and fed once a week.
Muscle Injury and Sampling
Animals were anesthetized with tricaine methane
sulfate (MS222), and pure cardiotoxin from Naja
mossambica nigricollis venom (Latoxan, France)
(10⫺5M in 0.9% NaCl) was injected into the right anterior brachial muscle of the forelimb (Nicolas et al.,
1996). We ascertained that intramuscular injection of
the solvent alone did not induce any deterioration of
the muscles. Samples for histological studies were
plunged in isopentane and frozen in liquid nitrogen.
For RT-PCR studies, samples were placed in cryotubes
and immediately frozen in liquid nitrogen. Before that,
to make sure of the completeness of the degeneration/
regeneration process, all the samples were analyzed in
transverse cryosections under the light microscope.
Muscle Denervation
Prior to cardiotoxin injury, the anterior brachial
muscle of adult Xenopus was denerved as follows. A
double proximal ligature and a double distal ligature
with silk thread, separated by 2–3 mm, were done one
the brachial nerve, which was then resected between
these ligatures.
Just after the cardiotoxin injection, animals were
treated with 3,5,3⬘-triiodothyronine (T3); water in the
breeding tanks was supplemented with T3 (5. 10⫺8 M),
and this medium was changed every day for two
Thyroid Hormone Assays
T3 serum concentrations were determined by a specific immunoassay (Immo Phase) modified from the
protocol recommended by Corning Medical Laboratories; in particular, the T3 antibody was diluted 10-fold.
Preparation and Prehybridization of Tissue
The procedure for fixing, embedding and sectioning
tissues was as for mouse embryos and was essentially
the same as described by Wilkinson et al. (1987) with
some modifications (Ontell et al., 1993). Briefly, tissues
were fixed in 4% paraformaldehyde in PBS, dehydrated
and infiltrated with paraffin. Then 6 ␮m thick, serial
sections were mounted on TESPA-coated RNase-free
glass slides. Sections were deparaffinized in xylene,
rehydrated, digested with proteinase K, postfixed,
treated with dithiothreitol/iodoacetamine/N-ethylmaleimide (to reduce non-specific 35S-binding; Zeller and
Rogers, 1989), treated with triethanolamine/acetic anhydride, washed and dehydrated.
Probe Preparation
The following probes were used to generate antisense
(a) XMyoD template is a 598 nt 3⬘ fragment (BamHIEcoRI; position 872-1469) of XMyoD2-24 (Hopwood
et al., 1989) subcloned in pGEM 4Z (Promega Biotec, Madison, WI), cut with BamHI and transcribed
with SP6 RNA polymerase.
(b) Xmyogenin template is a 522 nt fragment corresponding to a DNA sequence subcloned into
pGEM7Zf(⫹), plamid pCJMG2 (Jennings, 1992),
linearized with SphI and transcribed using SP6
RNA polymerase.
(c) pSP73-XMyf5-2 template (Hopwood et al., 1991),
was linearized using PstI and transcribed using
SP6 RNA polymerase. The RNA probe was a 534 nt
3⬘ fragment (PstI-EcoRI, position 601–1134).
(d) XMRF4, pCJM 4.2 (Jennings, 1992), was linearized
using BamHI and transcribed using T7 RNA polymerase. The RNA probe was a 3⬘ fragment
(BamHI-XhoI, position 721-3⬘ terminus).
cRNA probes were made by in vitro transcription in
the presence of 50 ␮Ci [35S]UTP at 1,200 Ci/mmol
(NEN research product), according to the manufacturer’s instructions (Promega Biotec, Madison, WI). However, unlabeled UTP was omitted from the reaction
medium in order to achieve synthesis of RNA probes
with a specific activity of 109 cpm/␮g.
Probes were hydrolyzed to an average of 100 nucleotides by limited alkaline hydrolysis, according to Cox
et al. (1984), for efficient hybridization, and used at
50,000 cpm/␮l hybridization solution. Thirty ␮l of hybridization solution was loaded per section.
Hybridization and Washing Procedures
High-stringency conditions for hybridization and
post-hybridization were followed. Sections were hybridized overnight at 53°C with post-hybridization
washing in 2⫻SSC, 50% formamide, 50 mM DTT at
65°C for 30 min. Autoradiography was carried out with
Kodak NTB-2 track emulsion, developed in Kodak D19
developer and stained lightly with Giemsa.
Evaluation of Hybridization Signal
In order to compare the intensity of hybridization
signal with a given cRNA probe over time, sections at
each stage were hybridized with the same probe preparation, washed, dipped into emulsion, exposed and
developed together. Changes in the level of hybridization signal with a given cRNA probe over time were
evaluated by taking dark-field micrographs, with a constant light intensity and a constant time of exposure of
the film to the light source.
For the histological results presented, transverse frozen sections, 8␮m thick, were used. They were stained
with hematoxylin and eosin.
RNA Extraction
Total RNA was purified by the method of Auffray and
Rougeon (1980) and was monitored for quality by agarose gel electrophoresis and ethidium bromide staining.
This was performed in one single tube, according to
Goblet and Whalen (1995) and Lin-Jones and Hauschka (1996).
First-Strand cDNA Synthesis
One microgram of total RNA was used for RT-PCR
for all primer pairs. The RNA was denatured briefly
with 25 ng of random primer (Gibco BRL) at 65°C for 5
min. Then 1X RT buffer (ATGC), 1 mM each dNTP, 10
U of RNasin (Promega) and 200 units AMV Reverse
Transcriptase (ATGC) were added to a final reaction
volume of 20 ␮l. Reverse transcription reactions were
incubated for 10 min at room temperature, 60 min at
37°C, and 5 min at 95°C.
PCR Amplification
The whole reverse transcription reaction was diluted
to 100 ␮l final volume with 1X PCR buffer (ATGC).
Multiplex PCR reaction was performed with a MRFspecific set of primers, and with a EF-1␣-specific sets of
primers (see below), and 2 units of Taq polymerase
(ATGC). Samples were overlaid with paraffin oil (80 ␮l)
and amplified in a thermocycler (Appligène, France).
The cycling parameters were as follows: the initial
cycle consisted of a 95°C denaturation for 5 min, 1 min
at a 55°C annealing temperature, and 1 min at a 72°C
extension temperature. The remaining cycles were for
30 sec at 95°C, 1 min at 55°C and 1 min at 72°C, with
the final cycle having a 10 min extension at 72°C.
Due to the different abundance of EF-1␣ and MRF
transcripts, we performed 9 PCR cycles with MyoD,
MRF4 or myogenin primer pairs or 12 PCR cycles with
Myf-5 primer pairs, before adding the EF-1␣ primers
and continuing amplification for 19 additional cycles.
One-fifth of the PCR sample was electrophoresed on
6% polyacrylamide gels. DNA was transferred onto
Hybond-C super membrane (Amersham). Southern
blots were hybridized with MRF and EF-1␣ radioactive
probes. The amount of EF-1␣ PCR product was quantified using a Bio-imaging analyzer and NIH image
analyzer software. This calibration allowed us to adjust
the volumes of the PCR reactions loaded on the polyacrylamide gel.
PCR Primers
(positions 524 –774, Hopwood et al., 1991). This primer
pair produced a fragment of 250 bp from cDNA.
(positions 662–952, Hopwood et al., 1989). This primer
pair produced a fragment of 289 bp from cDNA.
XMRF4 : 5⬘-CTTTTACCTGGATGGAG-3⬘ (F); 5⬘-TGGTGGAGCTAAGACAT-3⬘ (R)(positions 147–309; Jennings, 1992). This primer pair produced a fragment of
162 bp from cDNA.
(positions 35–183; Jennings, 1992). This primer pair produced a fragment of 147 bp from cDNA.
(positions 1088 –1311; Krieg et al., 1989). This primer
pair produced a fragment of 222 bp from cDNA.
Calibration of the RT-PCR Assay
In the first series of experiments, we used only one
primer pair per PCR and 1 ␮g of total RNA extracted
from regenerating muscles 15 days P-I. The total number of cycles varied from 5 to 40 and the exponential
step of the PCR reaction was determined.
In a second set of experiments we determined the
maximal RNA input by adding to the RT reaction serial
dilutions of total RNA extracted from regenerating
muscles 15 days P-I. The PCR was performed with
subsaturating cycle number.
In the latter experiments we determined the competitivity of the two primer pairs during the multiplex
PCRs with 1 ␮g of total RNA extracted from regenerating muscles 15 days P-I and a subsaturating number
of cycles. The rate of signal intensity obtained during
multiplex PCR was the same as the intensity proportion of separated PCR signals.
Negative Controls
Negative controls were performed with samples in
which the reverse transcriptase or RNA or one of the
primers was omitted, to detect eventual DNA contamination. All these controls remained consistently negative.
RNAse Protection Assay
RNAse protections were performed using the following probes: Xenopus AChR ␣ subunit template is an
EcoRI-BglII fragment corresponding to positions 237–
496 of the full-length SP65-AChR ␣ cDNA (Baldwin et
al., 1988). EF-1␣, G1EF.BS subunit template is a PstIBstEII fragment corresponding to positions 790 – 879 of
the full-length EF-1␣ cDNA (Krieg et al, 1989).
We thank Drs J.B. Gurdon, C.G.B. Jennings, P.A.
Krieg, and S.J. Burdon for the cDNAs. This work was
supported by grants from the Association Française
contre les Myopathies. Nathalie Nicolas held a doctoral
fellowship from the Ministère de la Recherche et de
l’Espace (MRE).
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